Many vaccines were generated and validated for preventing symptoms and mortality associated with viral infections of humans and animals. Some of these vaccines must be frequently updated because of the continuous genetic evolution of the viruses targeted, notably those vaccines aimed at preventing symptoms and mortality attributable to influenza A viruses. Such recurrent updating of the viral strains used to manufacture updated vaccines hamper production of large quantities of vaccines in due time. Moreover, the production costs of these recurrent updatings and the logistic chain that is necessary to ensure quick and wide distribution and administration of these vaccines frequently prevent the pig, chicken, turkey or equine industry from adequately vaccinating all the targeted animals in due time.
In humans, only adamantanes (amantadine, rimantadine) and inhibitors of neuraminidase (oseltamivir, zanamivir) are available for therapy of influenza A viruses-associated diseases. Large scale use of these molecules leads rapidly to emergence of resistant viral strains. As an example, most circulating influenza A virus strains are currently resistant to adamantanes and the prevalence of H5N1 strains resistant to inhibitors of neuraminidase increases constantly. As a consequence, chemotherapeutic molecules capable of mitigating influenza A virus-associated diseases in humans are very scarce.
In humans, the use of monoclonal antibodies to fight viral diseases was proposed. However, as these molecules comprise nonhuman segments, they often cause allergic reactions. Moreover, as many viruses are endowed with a very efficient genetic evolution capacity for evading new therapeutic molecules, the cost/benefit ratio expected from the process of developing a new antiviral monoclonal antibody dramatically hampers the discovery of new molecules.
In humans, gene therapy is an alternative approach to fight diseases as shown in the past for genetic diseases, cancer, or viral diseases (see PCT Publication Nos. WO91/02805, EP 0 415 731 and WO 90/07936). As vectors used in gene therapy transform only a fraction of host cells available for virus amplification, a credible antiviral transgene must encode for a very strong antiviral protein to give the gene therapy process any chance to attenuate the severity of the disease targeted. Such credible transgenes do not exist yet.
In animals, prevention of the spread of economically devastating or anthropozoonotic viral contagious diseases includes mass slaughtering. Today, this sanitary policy faces major economic and ethical concerns. Still in animals, it is theoretically possible to use genetically resistant genitors to disseminate resistance traits among progenies and, hence, progressively enhance the epidemiologic resistance of farm animal populations. However, this approach is based on prior identification of allelic variation at loci encoding for innate resistance against the disease targeted. Such projects are not realistic in terms of cost/success ratio, notably because many disease resistance traits are polygenic.
In 1962, the group of Lindenmann fortuitously discovered that the inbred mouse strain A2G spontaneously resisted experimental infections with influenza A viruses that were systematically fatal for other strains. The new resistance trait was noted Mx, standing for myxovirus resistance. Years after, it appeared that the Mx+ trait cosegregated with the expression, upon interferon alpha/beta (IFNα/β) treatment, of a ˜78 kDa protein that was henceforth named Mx protein. Since then, molecular genetic studies led to the identification of the genes underlying Mx proteins expression, first in mice, then in humans and subsequently in all vertebrate species studied. According to sequence homologies, vertebrate Mx proteins were shown to be large dynamin-like GTPases. Dynamins constitute a subfamily of high molecular weight GTPases that play critical functions in a large array of cell processes among which mobility, membrane remodeling, endocytosis, vesicular traffic and division of cell and organelles. Among dynamin molecules, some lack the typical pleckstrin and prolin/arginine-rich domains and their expression is subordinated to type I interferons; these are called “Mx” dynamins. Each vertebrate species possesses two or three Mx genes of which a few allelic versions were shown, in vitro, to encode for Mx dynamins endowed with antiviral activity, most often against influenza A viruses. Further researches revealed that some versions of Mx dynamins were endowed with antiviral properties and that various viruses were targeted, depending on the Mx isoform studied. Targeted mutagenesis studies later showed that the C-terminal GTPase Effector Domain (GED) of Mx dynamins supports antiviral activity and antiviral spectrum.
In the efforts to investigate antiviral activity the Bos taurus Mx1 dynamin sequence was made available. In vitro tests with cultured cells expressing bovine Mx1 gene revealed that human and bovine parainfluenza-3, human and bovine respiratory syncytial, bovine viral diarrhea/mucosal disease, Sendai, measles, and encephalomyocarditis viruses were not inhibited by the bovine Mx1, whereas vesicular stomatitis and rabies viruses were. Compared to other Mx dynamins, a specific antiviral spectrum was thus associated to the bovine Mx1 dynamin but as prior art had shown that other Mx dynamins display specific antiviral profiles, this was not unexpected.
With the notable exception of that encoded by the mouse Mx1+ allele, the prior art is deficient in Mx dynamins capable of suppressing influenza A viruses infection-associated diseases in vivo. As influenza A viruses constantly circulate in human, pig and poultry populations, it is trivial that human, porcine or chicken Mx proteins do not protect humans, pigs and chicken against severe, even fatal influenzal disease respectively. Using human, pig or chicken antiviral Mx1 dynamins for gene therapy is therefore not pertinent. Similarly, selection of genitors endowed with the best alleles, as determined in vitro, for raising progressively more resistant chicken or pig populations is not pertinent. Conversely, using the mouse Mx1 dynamin for gene therapy or for generating transgenic influenza-resistant food animals is theoretically pertinent. However, as mouse Mx1 dynamin is phylogenically distant from human Mx proteins, immunopathologic (allergic) problems may arouse from mouse Mx1-based gene therapies or from ingestion of mouse Mx1-containing food. Moreover, bringing murinized chicken, murinized turkey or murinized pig meat on the market would undoubtedly give rise to hostility of consumers. It is therefore highly desirable to use mutant human Mx dynamin with enhanced antiviral activity for gene therapy compositions. Similarly, it is highly desirable to create mutant food animal Mx dynamins with antiviral activity equal or superior to that exercised by mouse Mx1 in vivo in order to generate transgenic influenza-resistant food animals. The antiviral function of all anti-influenza Mx dynamins known so far is exercised through their C-terminal GTPase effector domain (GED).
TNF-receptor associated factors (TRAFs) form an array of adapter molecules that upon engagement of TNF-, IL-1β, TLRs and RANK receptors by their respective cognate ligands come first in contact with the activated receptor, acting as docking molecules for kinases and other effector proteins that are recruited to the activated receptor. TRAFs later regulate the subcellular relocalization of the receptor-ligand complex and modulate the nature and extent of the response by controlling the degradation of key proteins in the pathway. By doing so, TRAFs control activation of protein kinase cascades and transcription factors in the NF-kB and AP-1 families, thus tuning transcription of numerous genes that are involved in proliferation, differentiation and apoptosis.
The prior art is deficient in a method of inhibiting multiplication of viruses and/or to abolish or attenuate viral disease-associated cytokine responses and organ dysfunctions.
Therefore the object of the present invention was to provide animals, preferably transgenic animals which have a decreased susceptibility to influenza A virus. The object was further to provide medicaments for the preventive and or therapeutic treatment of influenza A virus-induced diseases.
Further, the object of the present invention therefore also was to provide a medicament for use in preventive and/or therapeutic treatment of influenza A virus, particularly for human use.